Genetic Polymorphism and Forensic Efficiency of Five X-STR Loci in - PDF document

Genetic Polymorphism and Forensic Efficiency of Five X-STR Loci in Korean Byung-Ki Kwon 1 , Kyoung-Jin Shin 1 , Sang-Seob Lee 1 , Gil-Ro Han 2 , Jong-Hoon Choi 1 , Chong-Youl Kim 1 1 Yonsei University 2 National Institute of Scientific Investigation Why X-STRs? � In deficiency cases, the mean exclusion chance of X-STRs tends to be higher then that of autosomal STR loci. � Males are hemizygous for X-STRs. � In some special cases of paternity testing , the established methods can be effectively supplemented by X chromosome marker investigation. 1

When are X-STRs applicable? � If in a kinship case the question to be solved is whether two women who were separated as children could be sisters , exclusion can be detected using X- markers in contrast to autosomal STRs. � X-STR may be valuable to conform a grandmother- grandchild relationship . � X chromosome marks are only applicable when the disputed child is female . Aim of this study � To explore 5 X-STR ’ s potential utility for forensic application. � GATA172D05 (Yuan et al . 1997) � HPRTB ( Edwards et al. 1991) � DXS8377 (Hu et al. 1996) � DXS101 (Allen and Belmont 1993) � HumARA ( Edwards et al. 1991) � Edelmann et al . Forensic Sci. Int. 2001 � Zarrabeitia et al . Forensic Sci. Int. & Int. J. Legal Med. 2002 2

Material and Methods DNA Extraction � Buccal swab samples were obtained from 150 unrelated Korean men and women. � Genomic DNA was extracted using QIAamp DNA Mini Kit (QIAGEN) according to the manufactures instructions. 3

Multiplex PCR Design 200 bp 300 bp 400 bp 100 bp GATA HPRTB DXS8377 DXS101 HumARA Primer of 5 X-STR loci Locus Sequence µM Dye GATA172D05 Primer 1 5’-TAG TGG TGA TGG TTG CAC AG-3’ FAM 0.05 Primer 2 5’-ATA ATT GAA AGC CCG GAT TC-3’ - HPRTB Primer 1 5’-TCT CTA TTT CCA TCT CTG TCT CC-3’ FAM 0.06 Primer 2 5’-TCA CCC CTG TCT ATG GTC TCG-3’ - DXS8377 Primer 1 5’-CAC TTC ATG GCT TAC CAC AG-3’ FAM 0.2 Primer 2 5’-GAC CTT TGG AAA GCT AGT GT-3’ - DXS101 Primer 1 5’-ACT CTA AAT CAG TCC AAA TAT CT-3’ HEX 0.4 Primer 2 5’-AAA TCA CTC CAT GGC ACA TGT AT-3’ - HumARA Primer 1 5’-TCC AGA ATC TGT TCC AGA GCG TGC-3’ HEX 0.8 Primer 2 5’-GCT GTG AAG GTT GCT GTT CCT CAT-3’ - 4

PCR Condition 10× PCR Buffer 1.7 ㎕ 95℃ 11 min dNTPs 1.0 ㎕ × 31 ~ 32 cycles Primer 2.0 ㎕ 94℃ 1 min Gold Taq Enzyme 0.3 ㎕ 55℃ 1 min Template DNA 0.5 ㎕ 72℃ 1 min dH 2 O 7.0 ㎕ --------------------- 60℃ 30 min Final Volume 12.5㎕ Allelic Ladder Construction � Fragment Analysis � ABI 310 Genetic Analyzer � GeneScan 3.1 � Allele Sequencing � BigDye Terminator Sequencing Kit 2.0 � ABI 310 Genetic Analyzer � Sequencing Analysis 3.3 � Sequence Navigator 1.0 5

Allele Designation � Allele Nomenclature � Generally followed the recommendation by the ISFG Commision. � HPRTB and HumARA used the allele nomenclature that appeared in previous studies. � Create GenoTyper Macro � GenoTyper 2.5 6

Statistical Analysis � PowerStat (Promega Co.) Heterozygosity � Polymorphic Information Content � � Desmarais et al. (1998) MEC (trio case) � PE (motherless case) � PD (male, female) � � Genetic Data Analysis (Lewis) Hardy-Weinberg Equilibrium � Linkage Disequilibrium � 7

Results and Discussion GATA172D05 Allele Female Male Cumulated 6 0.083 0.073 0.080 7 0.003 0.007 0.004 8 0.150 0.167 0.156 9 0.087 0.087 0.087 10 0.413 0.380 0.402 11 0.223 0.220 0.222 12 0.040 0.067 0.049 Het 0.733 - PIC 0.706 - MEC 0.706 - PE 0.596 - PD 0.898 0.762 8

HPRTB Allele Female Male Cumulated 11 0.030 0.047 0.036 12 0.323 0.293 0.313 13 0.420 0.407 0.416 14 0.193 0.180 0.189 15 0.027 0.053 0.036 16 0.007 0.020 0.011 Het 0.633 - PIC 0.621 - MEC 0.621 - PE 0.520 - PD 0.839 0.711 DXS8377 Allele Female Male Cumulated 40 0.003 - 0.002 41 0.007 0.013 0.009 42 0.043 0.013 0.033 43 0.030 0.027 0.029 44 0.083 0.100 0.089 45 0.110 0.113 0.111 46 0.150 0.087 0.129 47 0.087 0.133 0.102 48 0.120 0.167 0.136 49 0.130 0.073 0.111 50 0.077 0.080 0.078 51 0.057 0.080 0.064 52 0.047 0.027 0.040 53 0.023 0.033 0.027 54 0.007 0.040 0.018 55 0.013 0.007 0.011 56 0.007 - 0.004 57 0.003 0.007 0.004 58 0.003 - 0.002 Het 0.933 - PIC 0.897 - MEC 0.897 - PE 0.814 - PD 0.983 0.901 9

DXS101 Allele Female Male Cumulated 21 0.017 - 0.011 22 0.037 0.040 0.038 23 0.113 0.100 0.109 24 0.310 0.213 0.278 25 0.183 0.273 0.213 26 0.167 0.207 0.180 27 0.110 0.120 0.113 28 0.047 0.027 0.040 29 0.007 0.007 0.007 30 0.010 0.013 0.011 Het 0.827 - PIC 0.790 - MEC 0.790 - PE 0.662 - PD 0.942 0.810 HumARA Allele Female Male Cumulated 12 0.010 0.007 0.009 13 - 0.007 0.002 14 - - - 15 0.007 0.013 0.009 16 0.013 0.013 0.013 17 0.017 0.027 0.020 18 0.027 0.033 0.029 19 0.033 0.067 0.044 20 0.067 0.093 0.076 21 0.153 0.107 0.138 22 0.133 0.207 0.158 23 0.153 0.113 0.140 24 0.157 0.093 0.136 25 0.090 0.073 0.084 26 0.037 0.047 0.040 27 0.053 0.027 0.044 28 0.037 0.033 0.036 29 0.007 0.020 0.011 30 0.007 0.020 0.011 Het 0.847 - PIC 0.880 - MEC 0.880 - PE 0.812 - PD 0.978 0.899 10

HWE & Linkage Test � No deviation from Hardy-Weinberg equilibrium � No evidence of statistically significant linkage disequilibrium Mutation Rate of 5 X-STRs � HPRTB - 2 mutations among 580 meioses (Szibor et al . 2000) � DXS8377 - 1 mutation among 107 meioses (Zarrabeitia et al . 2002) � GATA172D05, DXS101, HumARA - No report on mutation rate 11

Mutation of DXS8377 � 1 mutation among 48 meioses 50 46, 49 49, 49 Conclusion � DXS8377 and HumARA which have many alleles are polymorphic STRs that can be very useful in forensic cases. � We believe 5 X-STRs work with reasonable amounts of DNA and may be particularly suitable for difficult deficiency paternity cases. 12